More than 170 million people worldwide are infected with the Hepatitis C Virus (HCV), a major human pathogen against which there is currently no vaccine and no sufficiently effective and tolerable therapeutic treatment available. In most cases, the infection causes chronic liver disease that often develops into cirrhosis and hepatocellular carcinoma. HCV is a small enveloped virus in the Hepacivirus is genus within the Flaviviridae family of positive-strand RNA viruses [2]. The viral genome is a messenger RNA of 9.5 kilobases, containing a single long open reading frame which is translated into a precursor polyprotein of ˜3010 amino acids. Maturation of the precursor into the individual viral proteins is carried out by cellular and viral proteases and takes place both co- and post-translationally [3]. The structural proteins are derived from the N-terminal portion of the precursor, and include the core (C) protein and the envelope glycoproteins, E1 and E2, arranged in this order from the N-terminus of the polyprotein.
Circulating HCV virions are associated with cellular components, in particular low- and very low-density lipoproteins (LDL and VLDL) [4], which results in heterogeneous infectious particles of low buoyant density. The virus targets essentially human hepatocytes, the entry process into which is not fully understood. A number of cellular entry factors (or putative receptors) have been identified, including the tetraspanins CD81 [5], Claudins 1, 6 and 9 [6,7], occludin [8], the scavenger receptor B1 (SR-B1) [9], the LDL receptor [10], and glycosaminoglycans (GAGs) [11]. The current data suggest that several of these cellular factors are recruited sequentially for virus entry [12], however the precise order and timing of the relevant interactions is not fully understood. The major players of the virion are the envelope proteins E1 and E2, but their individual specific roles during entry have not been experimentally demonstrated. It has been shown that after initial attachment to glycosaminoglycans [11] E2 binds to SR-BI, an interaction involving a segment called “hypervariable region 1” (HVR1) at the N-terminus of E2 [9, 12, 13]. Furthermore, E2 also interacts with CD81, the binding site of which includes three discontinuous stretches in E2 that are distant in the primary structure [14-17]. It has been reported that CD81 and SR-BI act cooperatively to initiate the entry process [18]. The HCV virion is then internalized by receptor-mediated endocytosis via clathrin-coated vesicles [19,20]. The low pH environment of the endosome is believed to trigger a fusogenic conformational change in the envelope proteins, inducing fusion of the viral and endosomal membranes and the release of the genomic RNA into the cytoplasm of the target cell.
The 3D organization of the HCV envelope has been poorly studied, essentially because of the difficulties in producing enough material for the relevant structural analyses. Several properties of the HCV envelope glycoproteins as well as of viral particles have therefore been inferred by extrapolation from better-studied members of the Flaviviridae family, namely the viruses forming the flavivirus genus. In spite of the lack of sequence conservation in the structural protein region, the members of the different genera within this family have the same genomic organization as HCV, encoding the structural proteins in the same order in the N-terminal portion of the precursor polyprotein. Moreover, the organization of the structural genes in HCV is also similar to members of the related Togaviridae family of small enveloped, positive-strand RNA viruses, comprising the alphaviruses genus for which structural studies are also available. Similar to HCV, the envelope proteins of viruses belonging to these families fold as a heterodimer in the ER of the infected cell and in both cases the first envelope protein has been shown to play a chaperone role in the folding of the second one [21,22].
The envelope proteins of flavi- and alphaviruses appear to have diverged from a distant common ancestor—as suggested by the crystal structure of their corresponding membrane fusion proteins, E and E1, respectively, which display the same 3D fold and are the prototype of the class II membrane fusogenic proteins. The acid pH induced fusogenic conformational changes of flavivirus E and alphavirus E1, have both been structurally characterized [23-25]. These structural studies have provided insight into the process of membrane fusion induced by the beta-rich class II fusion proteins, revealing important mechanistic similarities to that of the predominantly alpha-helical “class I” proteins (reviewed in [26]). It is widely believed that viruses belonging to other genera within these families—including HCV—are likely to code for class II fusion proteins as well. The tertiary structure of class II proteins features 3 distinct domains folded essentially as beta sheets, with a central domain I containing the N-terminus, a fusion domain II that is made from two polypeptide segments emanating from domain I, and a C-terminal domain III displaying an immunoglobulin superfamily fold located at the opposite side of domain I in the pre-fusion conformation. The conformational change leads to a trimerization during which the subunits adopt a hairpin conformation, bringing together the fusion loop and the trans-membrane segment, with domain III displaced by about 30-40 Å with respect to the other two domains, stabilizing the post-fusion homotrimer.
The similarities mentioned above have led to the proposal of a theoretical atomistic model of HCV E2 based on the class II fold, derived from the crystal structure of the flavivirus virus E protein homodimer [1]. This model was used to fit a low-resolution cryo-EM 3D reconstruction of HCV-like particles [27]. However, no experimental data supporting these models have been obtained so far.
Several studies have addressed the mechanism of membrane fusion initiated by the HCV glycoproteins [28-30], however the identity of the HCV fusion protein remains to be experimentally determined. Structural studies on E2 can provide important insights into its role during entry. Such studies can only come from the use of recombinant proteins, complemented by low resolution studies of authentic HCV virions. X-ray crystallography analyses on the individual proteins are however difficult, mainly because both E1 and E2 are heavily glycosylated [31]- and the presence of several glycans has been shown to be essential for folding in the ER lumen [32]. Their 3D fold is further stabilized by an important number of disulfide bridges—E1 and E2 display 8 and 18 strictly conserved cysteines, which are believed to be involved in 4 and 9 intramolecular disulfide bridges, respectively. These features concur to make production of the purified glycoproteins in sufficient quantities for structural studies a very difficult task.